Being HIV-infected is a stressful experience for many individuals. To assess HIV-related stress in the Chinese context, a measure with satisfied psychometric properties is yet underdeveloped. This study aimed to examine the psychometric characteristics of a simplified Chinese version of the HIV/AIDS Stress Scale (SS-HIV) among people living with HIV/AIDS in central China.st 2014 to August 31th 2015 by consecutive sampling. A standard questionnaire package containing the Chinese HIV/AIDS Stress Scale (CSS-HIV), the Chinese Patient Health Questionnaire-9 (PHQ-9), and the Chinese Generalized Anxiety Disorder Scale (GAD-7) were administered to all participants, and 38 of the participants were selected randomly to be re-tested in four weeks after the initial testing.A total of 667 people living with HIV  were recruited from March 1Our data supported that a revised 17-item CSS-HIV had adequate psychometric properties. It consisted of 3 factors: emotional stress (6 items), social stress (6 items) and instrumental stress (5 items). The overall Cronbach’s α was 0.906, and the test-retest reliability coefficient was 0.832. The revised CSS-HIV was significantly correlated with the number of HIV-related symptoms, as well as scores on the PHQ-9 and GAD-7, indicating acceptable concurrent validity.The 17-item Chinese version of the SS-HIV has potential research and clinical utility in identifying important stressors among the Chinese HIV-infected population and in understanding the effects of stress on adjustment to HIV. NationaBeing infected with HIV is an extremely stressful experience for many individuals, which affects almost every aspect of a person’s life , 7. A nuWith different operationalizations of the construct of stress, several established generic instruments or modified versions of these inventories  . PakenhaIllness is experienced within a cultural and social context which, in turn, can shape how people appraise aspects of their illness and how they cope with it . Hence, In 2003 Lin et al. , 29 tranIn this study, we aim to develop a culturally adapted and validated Chinese HIV-related Stress Scale (CSS-HIV), which would help to elucidate stress processes involved in HIV diagnosis, management, and treatment among people living with HIV in China. We also aim to explore the applicability of CSS-HIV among Chinese HIV-infected people, and shed light on its psychometric properties.Ethics approval was obtained through the Human Research Ethics Committee of Central South University, and written informed consent has been obtained from all participants.We used a cross-sectional design with consecutive sampling. Changsha City is the provincial capital of Hunan province in China. By the end of 2014, Hunan Province had 23,515 cumulated diagnosed HIV/AIDS cases, including 6,847 deceased cases, which represents the 7th highest number of cases of HIV/AIDS among all provincial districts in China. Sexual contact has become the primary mode of transmission in China, with 96% of new infections in Hunan province in 2014 being infected by sexual contact .st 2013 to August 31th 2014 a total of 667 people living with HIV who attended the free voluntary counseling and testing clinic of Changsha CDC and the Changsha Infectious Disease Hospital were recruited. Participants had to be aged ≥18 years old and living in Changsha City, including 6 districts and 3 counties. The response rate was 95.3% (667/700). All participants signed the informed consent form, and completed a questionnaire package. Thirty-eight participants were selected randomly to complete the questionnaire again in four weeks after the initial testing. The sampling procedure of this study has been described in detail elsewhere [During March 1lsewhere .Demographics such as gender, age, educational background, employment, and marriage status were collected by a purpose-built questionnaire. Medical and HIV related information was obtained from records in the HIV/AIDS registered management system, including mode of HIV-transmission, time since HIV-infection, CD4 count (/μl), and number of HIV-related symptoms as follows: persistent fevers for over 1 month; persistent diarrhea or watery stools for over 1 month; persistent cough for over 1 month; over 10% weight loss in the last 3 months; white patches in the mouth (thrush); recurrent herpes simplex; active tuberculosis; and others (indicated by participants).Distressing emotions related to HIV/AIDS also lists two examples, you feel angry or fearful; you feel anxious or depressed . The final Chinese version of SS-HIV was used in this study. Please see details of CSS-HIV in Approved by the first author of the SS-HIV (Pakenham ), a paneDiagnostic and Statistical Manual of Mental Disorder, Fourth Edition (DSM-IV) [Depression was measured by the PHQ-9, a 9-item screening instrument based on diagnostic criteria for major depression from the (DSM-IV) , 34. Eac(DSM-IV) . The PHQ(DSM-IV) , 36.GAD-7 is a screening instrument for anxiety, which consists of 7 items that reflect the symptom criteria for general anxiety disorder from the DSM-IV , 38. Res2/df <3, Comparative Fit Index (CFI)>0.90, Goodness of Fit Index (GFI)>0.90, Tucker-Lewis Index (TLI)>0.90, Root Mean Square Error of Approximation (RMSEA) <0.05 [Firstly, we conducted a confirmatory factor analysis (CFA) to examine the three-factor structures proposed by Pakenham and Rinaldis . The folA) <0.05 . The resA) <0.05 . After rInternal reliability and test-retest reliability was assessed using the criterion of Cronbach’s alpha ≥0.70 and the intra-class correlation coefficient (ICC) ≥0.70, respectively , 41. IntThe CFA analyses were performed with AMOS 7.0 . All the other analyses were performed with SPSS version 17.0 . Significance level of this study was set at 0.05.Of the 667 participants, 7.3% were female, and the mean age was 31.9 years with a range of 18 to 76 years (SD = 10.6). Most were single (62.7%), had attained tertiary education (46.3%), and had a stable job (56.2%). Most respondents (83.5%) were newly diagnosed (less than 1 month), and infected through sexual transmission (95.2%), in particular homosexual transmission (57.1%). Over one-third (36.7%) had HIV-related clinical symptoms, while 12.6% had a CD4 count lower than 200/μl.2 = 7126.713, P = 0.000), indicating the adequacy of proceeding with factor analysis. The EFA results indicated a three-factor model, but some items loaded on different factors rather than the original one. We conducted CFA on the revised 23-item three-factor model, which also showed a poor fit to the data with high χ2/df, CFI, GFI and TLI below 0.90, and RMSEA over 0.7.First, we conducted CFA on the original 23-item three-factor model, and poor levels of goodness of fit were found . Then weincreased drug/alcohol use) which was correlated with the total score <0.4 , and 85.5% endorsed “not at all”. EFA (with Varimax) and CFA were carried out on the retained 22 items, but the results still showed that the 22-item three-factor model was not ideal  with double loadings (>0.4) based on the EFA on 22 items, and conducted EFA (with Varimax) and CFA on the 17 items. The EFA yielded a three-factor solution that accounted for 56.274% of the variance, and the CFA goodness-of-fit measures supported the adequacy of this solution . Therefore, the 17-item three-factor structure was retained as the final model.Based on the EFA results of the 17-item CSS-HIV, Factor 1 consisted of 6 items representing “social stress”  and accounted for 40.532% of the variance; Factor 2 consisted of 6 items representing “emotional stress”  and accounted for 8.687% of the variance; whereas Factor 3 consisted of 5 items representing “instrumental stress”  and accounted for 7.054% of the variance. Eleven out of 17 items fell into the original SS-HIV factors, and item loadings ranged from 0.471 to 0.765 .Cronbach’s α for the 17-item CSS-HIV was 0.906 for the whole scale. As displayed in r = -0.104); and people infected through heterosexual transition tended to have lower stress than those infected through homosexual and other modes, especially emotional stress (r = 0.080). Gender, education and CD4 counts were unrelated to the CSS-HIV total and factor scores.As shown in In terms of depression and anxiety, the mean score of PHQ-9 was 78.09±6.63, and ranged from 0 to 27. 63.1% had depression symptoms (≥5) and 34.5% were classified in “moderate-severe” depression range (≥10). The mean GAD-7 score was 6.69±5.77, with a range from 0 to 21. The prevalence of anxiety was 58.6% (≥5), and 27.3% were classified with “moderate-severe” anxiety symptoms (≥10).The Spearman correlation analysis indicated that the total stress score, as well as the three subscales, were significantly and positively correlated with both depression and anxiety. The correlations were moderate, ranging from 0.510 to 0.711.This study presents the psychometric characteristics of a Chinese version of the HIV/AIDS Stress Scale (SS-HIV) in a representative sample of people living with HIV in central China. The results suggested that the 17-item CSS-HIV with three factors has adequate internal reliability, test-retest reliability and concurrent validity.concerns about death related to HIV/AIDS, difficulties in coming to terms with HIV/AIDS status, difficulty dealing with HIV-related symptoms or illness, and religious/existential difficulties related to HIV/AIDS. The fact that these items did not load unambiguously on the original emotional/existential stress dimension may reflect cultural differences in perceptions of these stressor and marked progress in the treatment and management of HIV/AIDS over the past 10 years. Regarding the latter, these stressors  are less likely to be prominent in the context of current relatively effective medical treatments, which have changed HIV/AIDS from being a life threatening illness to a chronic health condition.Item 10 which referred to substance abuse related to HIV/AIDS was removed. We found that most of the participants (86.7%) reported no increased drug/alcohol intake related to HIV/AIDS, although some prior research has shown that life stress is associated with increased substance use in people living with HIV . Meade’sr = 0.656). Different cultural contexts may alter the poignancy of the emotional or social elements of stressors represented in the items that changed between the social and emotional stress factors.Findings from the present study confirm the three-dimensional structure of HIV/AIDS related stress: social, emotional, and instrumental stress. In the 17-item three-factor structure of CSS-HIV, the majority of items loaded onto these original three factors. However, cultural differences in how some of the HIV/AIDS stressors in the CSS-HIV are experienced emerged. There were six items that loaded onto other factors rather than the original one, and the changes were mostly between social stress and emotional stress. According to Lazarus and Folkman’s (1984) conceptualization of stress and coping , the extSuicidal thoughts/attempts related to HIV/AIDS as example. This item originally loaded on the social stress factor, but in the present study it loaded on the emotional stress factor. There are various motivations for suicide. For instance, the interpersonal theory of suicide suggests that perceived burdensomeness and social alienation are the key elements that arouse one’s desire for death [Take the item or death . In Shneor death , a desiror death –48. ThesThe reliability findings supported that the 17-item CSS-HIV and its three subscales have good internal consistency (Cronbach’s α 0.791–0.906), which were similar to findings of the original 23-item scale (0.76–0.85), but better than the 23-item Chinese versions developed by Lin et al. (0.55–0.73)  and Gao r = 0.661 for PHQ-9 and 0.678 for GAD-7; p<0.001) and, thus, the construct validity of this 17-item scale was supported, and demonstrates the expected link between HIV/AIDS related stress and depression and anxiety.Consistent with Pakenham and Rinaldis’ study , this stincreased drug/alcohol intake, which might be a prominent stressor for HIV-infected IDUs, was the least stressful event in our sample where only 1.2% were IDUs. Another limitation is the lack of representation of people with longer time of infection and advanced courses of AIDS, and this may have produced a bias towards lower levels of stress. Future research on the CSS-HIV should be conducted with other Chinese HIV-infected groups, and continue to evaluate its psychometric properties.The main limitation of this study is the sample characteristics. Most of the participants were male and more than half of them were infected via homosexual transmission. HIV-infected sub-populations, such as injecting drug users (IDU), female sex workers and pregnant women may have different stressful experiences due to HIV-infection. For example, Item 10 refers to In conclusion, results from the present study show that the 17-item CSS-HIV is a psychometrically sound instrument for measuring HIV/AIDS-related stress among people living with HIV in China. The CSS-HIV shows potential in identifying people living with HIV who may be at risk for mental health problems. The CSS-HIV is also useful in identifying important stressors among HIV-infected Chinese, which will help to inform the development of targeted stress management programs. Having a common scale that assesses HIV-related stress will also assist research that explores comparisons in HIV across countries.S1 Appendix(PDF)Click here for additional data file.


Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate , or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as  Mycobacterium bovis BCG is currently the world's most widely used vaccine and has been given to more than three billion people, making it a very attractive prospect for the development of a live recombinant BCG (rBCG) multivaccine (–Hsp60 from BCG and PHsp70 from M. tuberculosis), the IS900 open reading frame promoter (PAN from M. paratuberculosis), and the mutated β-lactamase promoter (PBlaF* from M. fortuitum) are the ones used most, as reviewed in reference M. tuberculosis antigen 85B (Ag85B) in rBCG was placed under the control of a limited set of promoters found that increasing promoter activity caused a skewing of the immune response to Ag85B in mice from a mixed Th1/Th2 response to a predominantly Th1 response  and 5′-TAGATCGATCAGCTGTTACTTGTACAGCTCGT-3′ . This fragment was cloned into pJH152  between the KpnI and PvuII restriction sites, thus generating pJH-egfp (GTTTAAACAAACGGAAACAGCTATGACCAT-3′ (the PmeI restriction site is in bold) and 5′-TAGCATATGCGATCTCCCTTTCCCGT-3′ (the NdeI restriction site is in bold). Plasmid pJH-egfp and the PCR product were digested with PmeI and NdeI, resolved in an agarose gel electrophoresis to remove the original cassette and clean up the PCR product, and further purified with the QIAquick PCR Purification kit . Before ligation, the digested vector was treated with calf intestinal alkaline phosphatase (Promega) in accordance with the manufacturer's instructions for the dephosphorylation of 5′ overhangs and purified once again. Digested fragments were allowed to ligate with T4 DNA ligase (Promega) at 16°C for 16 h and used to transform E. coli DH5α. Plasmid DNA was extracted from approximately 2,000 colonies in a pool, generating a pJK plasmid library that, in turn, was used in the electroporation of M. smegmatis MC2155 with a Bio-Rad Gene Pulser II apparatus . Transformants were allowed to grow for 3 to 5 days on MB7H10-kanamycin plates  set up for excitation and emission wavelengths of 485 and 535 nm, respectively. Also, the optical density at 620 nm (OD620) of each sample was measured. The fluorescence of the strains was initially screened in fluorescence units (FU), and then, following normalization by OD, five strains were selected on the basis of measurements of relative FU (RFU). The plasmid in each of these transformants was extracted with the illustra plasmidPrep Mini Spin kit , and PL5 promoters were sequenced with the primer 5′-TCAGCTTGCCGTAGGTGGCA-3′.After visualization of M. smegmatis/pJK in triplicate to an OD of ∼0.1. Culture turbidity and fluorescence were monitored every hour. For comparison, wild-type M. smegmatis strain MC2155 grown in MB7H9 was also analyzed. A FACSCanto II flow cytometer  was used to determine the single-cell fluorescence of gated bacterial cells on the basis of the median of fluorescein isothiocyanate . Turbidity was measured with an Ultrospec 2000 spectrophotometer . To compare the newly generated promoters with a widely used one, PHsp60 was inserted in place of PL5. M. bovis BCG bacteria containing these plasmids were analyzed and compared with M. smegmatis constructs. Late-log-phase cultures of recombinant M. smegmatis strains were disrupted on ice with a GE 100 Ultrasonic Processor at half-maximum constant output, and the protein concentration in culture lysates was determined by the DC Protein Assay (Bio-Rad) with bovine serum albumin as the standard. Approximately 10 μg of soluble protein extracts was separated by SDS-PAGE and stained with Coomassie brilliant blue (Bio-Rad). The predicted molecular mass of GFP is 28.3 kDa (http://web.expasy.org/compute_pi/). Following destaining, the band corresponding to GFP's molecular weight was analyzed by densitometry with ImageJ software.Shaking flasks with MB7H9-kanamycin were inoculated with selected M. smegmatis/pJK strains were observed under a microscope. Late-log-phase cultures were washed twice with phosphate-buffered saline and placed on microscope slides. Images were acquired on an LSM 510 META confocal system  with a 488-nm laser for excitation and a 500- to 550-nm bandpass filter for emission.egfp gene by digestion with NdeI and PvuII and cloned the partial sequence of the Schistosoma mansoni gene sm29 (GenBank accession no. AF029222) codon optimized for expression in mycobacteria, as this antigen is currently considered a vaccine candidate . BCG was transformed with these constructs and allowed to grow as described previously. Kanamycin-resistant clones were grown until late log phase, disrupted on ice, and quantified as previously described. Approximately 25 μg of total protein extracts was separated by SDS-PAGE, and proteins were electrotransferred to a polyvinylidene difluoride membrane  and blocked for 16 h at 4°C in a 5% nonfat dry milk solution. Western blotting of Sm29 expression used polyclonal mouse anti-rSm29 antibody  and a horseradish peroxidase (HRP)-conjugated anti-mouse antibody adsorbed to human serum proteins  at 1:3,000. The chemiluminescent signal was detected with the Immobilon Western Chemiluminescent HRP Substrate , and images were acquired with the LAS4000 digital imaging system (GE).To assess the usefulness of the pJK library regardless of the downstream gene under its regulation, we used three plasmids containing the mutagenized promoters, pJK-F8, pJK-B7, and pJK-C1. For this model, we removed the andidate , creatinE. coli BL21(DE3) (Invitrogen) transformed with pET-21 containing the Sm29 cDNA sequence and purified by metal affinity chromatography as previously described  and Tukey's posttest. Correlation of interspecies fluorescence was tested with a nonparametric Spearman test. Growth curves were compared by nonlinear regression.For statistical analysis, the GraphPad Prism 5 software  was used. The relationship between pJH-L5 promoters and assess the strengths of variant sequences by cloning the promoters to drive enhanced green fluorescent protein (eGFP) expression . M. smegmatis/pJK-C1 was the only strain whose growth was demonstrated to be significantly different from that of the wild-type strain .On the basis of the initial screening, five strains covering the fluorescence range were selected for further analysis. First, the sequencing of selected promoters revealed multiple mutations with modification in 6 to 11% of their sequences . Since td in RFU . Likewisd in RFU . To exclM. bovis BCG, and late-log-phase cultures were analyzed alongside recombinant M. smegmatis by flow cytometry. After gating of the bacilli's population, fluorescence was determined as the median FITC value. M. smegmatis transformed with pJK-F8 showed high fluorescence; M. smegmatis transformed with pJK-B7, pJK-E6, and pJK-D6 showed intermediate fluorescence; and M. smegmatis transformed with pJK-C1 showed strikingly low fluorescence  according to the strength of each promoter. BCG transformed with the same plasmids revealed a high correlation with the observed fluorescence of M. smegmatis  (not shown), where all of the mutants of both species maintained their high, intermediate, or low strength. pJHsp60 demonstrated a strength comparable to that of the lowest-expression vector, pJK-C1  than with the reaction in the rBCG/pJK-C1.Sm29 extracts (lower promoter activity) at the same molecular weight as recombinant protein rSm29, but no immunoreaction was observed in wild-type BCG protein extracts . It could be argued that fluorescence may not serve as a good parameter to determine protein expression and ultimately promoter activity. However, here we have demonstrated that fluorescence determined by flow cytometry correlates with the total amount of GFP expression in SDS-PAGE even though only six promoters were used.The observed 6 to 11% mutations in the regulatory sequence is consistent with the expected rate of 10 to 15% mutagenesis with equimolar concentrations of altered and natural dNTPs . A higheIn this study, we observed mutations throughout the whole sequence, including the −10 and −35 regions of the promoters, exhibiting high, medium, or low strength. Interestingly, all of the midstrength promoters  showed a mutation at the first position of the −10 region (C → T). Also, the weakest promoter that we identified, in pJK-C1, is the only one with a mutation at the predicted transcriptional start site (A → G).M. smegmatis and M. fortuitum have a doubling time of 2 to 3 h and M. bovis BCG and M. tuberculosis have a doubling time of 20 to 24 h , an intermediate (pJK-B7), or a weak (pJK-C1) mutant promoter. rBCG expressed Sm29 at different levels, which corresponded to their eGFP-measured promoter strength. pJK-F8 was not evaluated since no colonies of rBCG were obtained, even after several attempts.To evaluate the utility of PrBCG/pJK-B7 clones were 9-fold more fluorescent than rBCG/pJK-C1 clones . To achieve such a difference, Bourn et al. used an enrichment method to select for high-copy-number variants of plasmid pORI101 with GFP expression 7-fold higher than that of the original . Althougegfp and pJK is derived from pAL5000 of M. fortuitum, which is widely used in mycobacterial shuttle plasmids such as pMV261, pLA71, and pMIP12. Its copy number in M. smegmatis was determined as 3 by Southern blot assay (M. smegmatis, the fluorescence values do not intersect each other.Since pJK plasmids are extrachromosomal, their copy number could be partially responsible for the differences in fluorescence or Sm29 expression observed among mycobacterial hosts. The origin of replication of pJH-ot assay , 8 by anot assay , and 23 ot assay . The varIn conclusion, this study demonstrates the utility of the rational design of systems to obtain a range of expression of target proteins in mycobacteria. The systems described present an alternative way to obtain the expression of foreign genes in mycobacteria and to design a new generation of rBCG vaccines that optimize the host immune response for protection.


Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice.An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR  with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting  Alicyclobacillus spp. are one of the most important quality and safety factors in acidic food products [Alicyclobacillus spp. in fruit juices can cause sour spoilage [Alicyclobacillus spp. have been isolated from apple, kiwi, grapefruit, orange and many kinks of fruit products and have posed a major challenge to the fruit juice and beverage industry [Alicyclobacillus spp. in fruit juice is critical.products , 2. The spoilage –5. The mspoilage , 7. At pindustry –11. The Alicyclobacillus spp. in fruit juice [The traditional culture methods for target bacteria detection are labor-intensive, time-consuming and inefficient, so they cannot guarantee the absence of it juice –14. As ait juice –17. SYBRit juice –21. And it juice , 22, 23.it juice –26. In tit juice , 28.Alicyclobacillus spp. in kiwi fruit juice. The target 16S rDNA sequences were identified and the primers set suitable for the real-time PCR system was developed. The assay for the identification and quantification of Alicyclobacillus spp. in fruit juice using IMS-real-time PCR was established. The correlation coefficient, detection limit, repeatability and amplification efficiency were evaluated.The aim of this study was to use the IMS test plus a SYBR Green I real-time PCR assay  for detection of Alicyclobacillus spp. and 5 Bacillus spp. were purchased from German Resource Centre for Biological Material (DSMZ). The other twenty-two wild-type isolates were isolated from apple or kiwi fruit orchard or fruit juice production line and obtained from microbiology laboratory of the College of Food Science and Engineering, Northwest A&F University, Yangling, China [The strains used in this study are listed in g, China , 29, 30.The extraction and quantitation of DNA were conducted as our previously reported . Cells wFor detection, the samples were treated according to our previously developed immunocapture procedure , 31. FivA. acidoterrestris , A. contaminans (DSM-17975), A. acidocaldarius , A. fastidiosus (DSM-17978), A. herbarius (DSM-13609), A. hesperidum (DSM-12489), A. sendaiensis (DSM-17614), A. acidiphilus (DSM-14558) and A. pomorum (DSM-14955) were obtained from GenBank . Then these sequences were analyzed and aligned for primers design using software Primer Premier 6 . A further computer-based specificity search was conducted by Clone manager suite 7.0 to confirm the specificity of designed primers. In this way, a short amplicon (188 bp) was chosen for real-time PCR analysis aiming to detect Alicyclobacillus spp.. Two conserved oligonucleotides (the forward primer 5’-ATGCGTAGATATGTGGAGGA-3’ and the reverse primer 5’- CAGG CGGAGTGCTTATTG-3’) were derived; the melting temperature (Tm) was 51.1°C and the length of them were 20 and 18 bases, respectively. After that, the primers were synthesized at TaKaRa Biotechnology Co., Ltd., Dalian, China, and the specificity was confirmed by PCR amplification with the parameters described below.For real-time PCR primer design, DNA sequences (16S rDNA) of Alicyclobacillus spp. DNA in biological samples was developed. This procedure was carried out in final reaction volumes of 25 μL with 12.5 μL of SYBR Green I Premix Ex-TaqTM , 0.5 μL of each primer set (10 μM), 1.0 μL of template DNA and 10.5 μL of ddH2O. Thermal cycling was performed on the iCycler-iQ5 thermal cycler  using the cycling conditions: pre-denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min. The amplification program was then followed by a melting cycle of 95°C for 0 s, 60°C for 30 s, and slow heating to 95°C for 0 s, with a transition rate of 0.5°C/s. The real-time PCR amplification of the targeted DNA was monitored by the increase of the fluorescence in real time. The positive amplification of 16S rRNA gene was determined when the PCR cycle crossed the threshold cycle (CT) value and confirmed by the DNA melt curve analysis.A real-time PCR assay for detection and quantification of Alicyclobacillus spp. and Bacillus spp. were used as templates  with a concentration of 2.8×107 CFU/mL was subjected to a 10-fold serial dilution in sterile water. The extracted DNA was used as template for the real-time PCR amplification. Each dilution was carried out three times in triplicate and operated by real-time PCR.The specificity of the real-time PCR was determined by comparing results when different DNA samples of emplates . The culA. acidoterrestris (DSM-3923) was diluted tenfold in kiwi fruit juice, with the cell concentrations ranging from 2.8×10−1 CFU/mL to 2.8×106 CFU/mL. The bacteria diluted by sterile water were considered as control. Then these prepared samples were immunocaptured by IMPs and detected by the real-time PCR assay. The samples without IMS enrichment were also considered as a control and tested by the same approach. At the same time, the obtained data were analyzed statistically and the reproducibility of the results was assessed. Intra-assay and inter-assay variability was determined according to MIQE  guidelines [The artificially contaminated kiwi fruit juice was prepared as the spiked samples and the performance of the developed IMS-real-time PCR assay was evaluated. The culture solution of idelines .A total of 86 natural kiwi fruit juice samples were used to evaluate the effectiveness of the IMS-real-time PCR. Sixty-nine of them was obtained from the kiwi fruit juice production line of Yang ling global horticulture Co., Ltd, and the other 17 samples within the quality guarantee period were purchased from the local supermarkets in Yangling, Shaanxi, China in 2014. For detection, the soluble solid content of samples was adjusted below 15°Brix and then these samples were treated according to our previously developed immunocapture procedure. Then the genomic DNA was obtained and detected by the established real-time PCR. The samples were also detected by a standardized pour plating method issued by the Japan Fruit Juice Association and the results were compared. The sensitivity, specificity and accuracy of the IMS-real-time PCR procedure were also evaluated , 34.A. acidoterrestris (DSM 3923) cultivar following the method described by our previously report [CT values as a function of the logarithm of known spiked quantities and determined in triplicate. The correlation coefficient (R2) was calculated and used to verify the fit of the trend line to the points on the curve. The amplification efficiency (E) was estimated by the formula E = 10−1/s-1, where s is the slope of the standard curve [CT plots, it was possible to quantify samples with unknown amounts of Alicyclobacillus spp.. Each experiment was carried out three times in triplicate.Standard curves were obtained for the y report . The currd curve . From thAlicyclobacillus spp. and sixteen Bacillus spp. were used as representative strains to evaluate the specificity of the real-time PCR assay. For detection, the genomic DNA of each strain was extracted as template and the DNA concentration was about 20 ng/μL. The representative real-time PCR amplification curves were presented in Alicyclobacillus spp. were positive in the real-time PCR with the mean CT values of 26.0 ± 1.0 and the non- Alicyclobacillus spp. had no amplification of DNA. To verify the specificity of the reactions using SYBR Green I as a fluorescent dye, melting curve analysis was performed and a reproducible distinct melting point of 80.5°C was observed for all Alicyclobacillus spp. amplicons  were used as template to evaluate the sensitivity of the developed system. As seen in 2 CFU/mL. The curve showed linear regression relationship with coefficient of determination (R2) of 0.9854 and a slope of Y = −3.2263X + 42.274 corresponding to the amplification efficiency of 104.2%  were regarded as negative in this study. In addition, only one single sharp peak at 80.5°C was visible in the melt peak chart of 10-fold serial dilutions of tested cells and there was no primer-dimers and non-specific product (2 CFU/mL of Alicyclobacillus spp.The genomic DNA obtained from f 104.2% . The res product . Based oA. acidoterrestris (DSM-3923) from the artificially contaminated kiwi fruit juice. It was found that the established method allowed the quantification of target strains in the linear dynamic range of 101−106 CFU/mL. The detection limit of the real-time PCR assay was improved one order of magnitude and that was 2.8×101 CFU/mL. The correlation coefficient obtained from the standard curves were greater than 0.986, and the amplification efficiencies of kiwi fruit juice and sterile water samples were 98.9% and 101.8%, respectively. With the pre-treatment of IMS technique, the background interference of the juice ingredients had little influence on detection of Alicyclobacillus spp.. The amplification efficiencies and detection limit obtained from fruit juice sample were consistent with the results from sterile water. In addition, the reproducibility of the developed assays was evaluated based on the CT values obtained from three separate reactions within each run (intra-assay) or in three different runs (inter-assay). For each dilution, the mean, standard deviation (SD) and coefficient of variation (CV) were calculated based on the CT values (Alicyclobacillus spp. in fruit juice.To improve the performance of the developed assay, IMS was used for isolation and enrichment of T values . The calAlicyclobacillus spp. negative by IMS-real-time PCR was positive using YSG agar. Three samples displayed positive by IMS-real-time PCR, but was Alicyclobacillus spp. negative by plating culture method. It showed that the IMS-real-time PCR procedure and pour plating method were consistent for detection of Alicyclobacillus spp. in 82 fruit juice samples. The sensitivity, specificity and accuracy of the developed assay were 91.7%, 95.9% and 95.3%, respectively. The results indicated that the proposed IMS-real-time PCR could be effectively used for the detection of Alicyclobacillus spp. in fruit juice.In this study, 86 naturally contaminated kiwi fruit juice samples were detected by the IMS-real-time PCR and standardized pour plating method. As shown in Alicyclobacillus spp. are among the most important spoilage bacteria in fruit juice. Since they were first isolated in 1984, Alicyclobacillus spp. have become a major concern to the global juice and beverage industries [Alicyclobacillus spp. in fruit juice and beverage is urgently needed.dustries , 9. The Alicyclobacillus spp. in fruit juice.With the advantages of less expensive, simple in primer design and universal real-time-PCR protocols suitable for target sequences, SYBR Green I based real-time PCR can be applied directly to any gene without the need to design and synthesize fluorescently labeled target specific probes , 21, 36.Alicyclobacillus spp. by IMS-real-time PCR. In our previous study, an immunocapture procedure has been established for separation and enrichment of Alicyclobacillus spp.. The ELISA or PCR was combined with IMS to shorten the total analysis time and improve the sensitivity for detection of Alicyclobacillus spp.. These methods contribute to the qualitative detection and differentiation of target strains in an efficient way [Alicyclobacillus spp.. The correlation coefficient of the standard curves was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%-101.8%. The detection limit of the IMS-real-time PCR assay was 2.8×101 CFU/mL. The sensitivity, specificity and accuracy of the testing system were 91.7%, 95.9% and 95.3%, respectively. The results showed that the IMS-real-time PCR procedure could be potentially useful for detecting Alicyclobacillus spp. in fruit products.As far as we know, this is the first report on the detection of ient way , 33. In Alicyclobacillus spp. in kiwi fruit juice. The established method allowed the quantification of target strains in the linear dynamic range of 101−106 CFU/mL with high correlation and PCR efficiency. Due to its high throughput nature, this approach is convenient for identification of target strains from different fruit juice samples. Further, this assay can be considered as an alternative to the regular methods of Alicyclobacillus spp. detection such as plating culture, conventional PCR and ELISA.In conclusion, a rapid and sensitive IMS-real-time PCR assay was developed for detection of


AGT; angiotensin-converting enzyme, ACE; angiotensin II type 1 receptor, AT1R) in HT and non-HT subjects were included that showed no significant genotype differences. In 2- to 6-locus models of the SNP-SNP interaction, the SNPs of AGT and ACE genes were associated with hypertension  1.26~6.21; P < 0.005). In 7- and 8-locus model, SNP A1166C of AT1R gene is joined to improve the maximum Boot-OR values of 4.050 to 4.483; CI = 2.49 to 7.29; P < 1.63E − 08. In conclusion, the epistasis networks are identified by eight SNP-SNP interaction models. AGT, ACE, and AT1R genes have overall effects with susceptibility to hypertension, where the SNPs of ACE have a mainly hypertension-associated effect and show an interacting effect to SNPs of AGT and AT1R genes.Several single nucleotide polymorphisms (SNPs) of renin-angiotensin system (RAS) genes are associated with hypertension (HT) but most of them are focusing on single locus effects. Here, we introduce an unbalanced function based on multifactor dimensionality reduction (MDR) for multiloci genotypes to detect high order gene-gene (SNP-SNP) interaction in unbalanced cases and controls of HT data. Eight SNPs of three RAS genes (angiotensinogen,  RAS genes such as angiotensinogen (AGT) [ACE) [AT1R) [ AGT gene but not the SNPs A-6G and M235T of AGT gene may associate with hypertension in patients from Taiwan [ ACE gene and +1166 C allele of AT1R gene are reportedly associated with hypertension [The renin-angiotensin system (RAS) represents a critical endocrine regulator for maintaining blood pressure and blood fluid volume in the circulatory system. Single nucleotide polymorphisms (SNPs) ofen (AGT) , 2, angiT) [ACE) , 4, and [AT1R) , 6 are k[AT1R) –9. For ertension . However AT1R gene and I/D of ACE gene have synergistic effects on acute myocardial infarction [ AGT gene, I/D of ACE gene, and A1166C of AT1R gene have been examined in coronary artery disease [ AGT gene and I/D of ACE gene. Additionally, joint effects of gene-gene interactions were discovered in blood pressure regulation [Accumulating evidence indicates that high order gene-gene (SNP-SNP) interaction can deeply affect disease susceptibility. For example, the A1166C of disease . A signigulation , left vegulation , and acuTo date, several computational methodologies have been proposed to detect the epistasis in many association studies –22. Data AGT gene, I/D of ACE gene, and A1166C of AT1R gene. We aimed to find out the influence of these 8 SNPs on hypertension outcomes. The results show that unbalanced function based MDR can avoid the drawback of common MDR in an unbalanced real data set. Thus, the best unbalanced function based MDR model can correctly predict high order SNP-SNP interactions of hypertension susceptibility using real data sets.Here, we describe a case-control study of hypertension susceptibility that specifically evaluates gene-gene interactions using unbalanced function based MDR  that comThe MDR method was briefly introduced and the unbalanced function based MDR method was explained in detail as follows.In 2001, Ritchie et al. proposed a MDR to detect the potential gene-gene interaction. MDR is a robust nonparametric method that detects nonlinear interactions among multiple discrete genetic factors . It is aDivide the data set into 10 subsets for cross-validation (CV).ith data set as the testing data and others are the training data.Keep the Calculate the total number of cases and the total number of controls within each multifactor class.n × n grid).Evaluate the ratio between cases and controls in each genotype combination /low (L) risks in each multifactor class. If the cases/controls ratio particular threshold, it is labeled with “H”; otherwise it is labeled with “L”.Compute the four frequencies of true positive (TP), false positive (FP), true negative (TN), and false negative (FN) in a 2-way contingency table.Evaluate the misclassification error.Repeat for each combination.Select the best model according to minimum misclassification error and record it into cross-validation consistency (CVC).Repeat for each CV interval.Select the best model according to the model with the highest frequency in CVC.n loci and a possible multiloci class are represented in the following n-dimensional space:n is designated depending on the number of factors being considered. Then, a set of n genetic factors is selected. The total numbers of cases or controls are counted in the multifactor class, and the ratio of the numbers of cases to controls is calculated. From ; otherwise, label is “L” (low-risk group). The threshold is equal to the one in a balanced data set. Thus, the huge genotype combinations in n-loci are reduced into a 2-way contingency table  and allow the statistical analysis to evaluate the n-loci effect. MDR uses the misclassification error to evaluate a model value where the misclassification error function is calculated as follows:Next, the high/low risk in each multifactor class is determined. Each multifactor class in i-fold. The training model is then used to test the testing data and record the TP, FP, FN, and TN for evaluating the statistical power. Thus, MDR repeats the above procedure in each CV interval. The MDR model with the lowest number of misclassified individuals is chosen and ten best models are classified by the same combination in the CVC. Finally, the highest occurring frequency in CVC is considered the best model. If a tie between 2 or more models happens, then the first appearing model is considered the best model.After all the multifactor combinations are evaluated by misclassification error, the MDR model with the minimum error rate individual is chosen and the model is considered the best model of training data at n × n grid for cases) to the percentages of controls in each genotype combination of control data  is proposed to classify the data to the high- and low-risk groups. Thus, the highest ratio between case and control groups can be clearly detected. The strategy is to modify the ratio between cases and controls in the ratio function of MDR, that is,  if all parameters l in vector L match their cases or controls; a mismatch is given the score “0.”The function Our strategy is to modify the misclassification error rate function of MDR, that is, . Equatiohttp://dx.doi.org/10.1155/2015/454091).Here we provide an example to show how the unbalanced function based MDR works .The power statistical analysis was implemented by the G1.5 tool , 29. Then = 313) and nonhypertension (n = 130) was obtained from our previous study [http://bioinfo.kmu.edu.tw/non-HT_and_HT_genotype_data.xlsx. Eight SNPs were included: T174M (rs4762), M235T (rs699), G-6A (rs5051), G-217A (rs5049), G-152A (rs11568020), A-20C (rs5050), I/D (rs4646994), and A1166C (rs5186) of three RAS genes . However, the possible SNP-SNP interaction was not examined. Here, we used the unbalanced function based MDR with minimum misclassification error rate to identify the best SNP-SNP interaction model with significant differences between hypertension  and nonhypertension  groups.The hypertension data set with hypertension  of each individual SNP. Among these eight SNPs, most individual SNPs paired with any genotype show no significant difference (P > 0.05) between the HT and non-HT groups. The frequency difference of SNP I/D of ACE gene between HT and non-HT groups is significant when based on chi-square test . However, it is not significant after a Bonferroni correction .P values, the best model was selected according to the model with the highest frequency in CVC which consisted of the model with the lowest error rate in each CV. Among these models, the lowest error of the best model is 0.419 for a 2-locus model (AGT G-217A + ACE I/D). Similarly, all the best models in 3- to 8-locus models are listed in All significant 2-locus SNP-SNP interactions  are know AGT and ACE genes were associated with hypertension. Both 7-locus and 8-locus models suggest that the listed SNPs in AGT, ACE, and AT1R genes were important in association with hypertension. Additionally, power analysis represents the degree of rejection for H0 that is significant at α = 0.05. Applying the MDR method, the testing data results were always not significant (at P > 0.05). Thus, we defined H0 as the result of the test set is the same as for the training set (H0), and H1 shows that the result of the test set was different from the training set. The powers in 2- to 8-locus, ranging from 0.901 to 0.999, indicate that occurrence probabilities in all models are higher than 0.9. These findings suggest that all these 8 SNPs are significantly associated with hypertension.P < 0.01, data not shown). The OR values in 2- to 8-locus models increase from 2.054 to 4.628. For the implementation of bootstrapping in 1000 samples, the adjusted OR (Boot-OR) values increase from 1.972 to 4.483 in 2- to 8-locus models and the P values of 2- to 8-locus models decrease from 0.003 to 1.48E-09. Both OR and Boot-OR values gradually increase when the loci numbers increase indicating that the hypertension risk is increasingly raised by the joint effect of SNPs. It also suggests the SNPs of AGT, ACE, and AT1R genes are highly associated with hypertension risk. SNPs G-217A (AGT) and I/D (ACE) occur in all best models of 2 to 8 loci in association with hypertension. The associated effects of A1166C (AT1R) are detected at the best 7- and 8-locus models and this leads to the highest risk compared to other models.In ACE), is significantly associated with hypertension using the chi-square test. However, it is nonsignificant after Bonferroni's correction  of MDR  and controls (n = 130) whereas In contrast, we demonstrated that our proposed unbalanced function based MDR is suitable for an imbalanced data set. For example,  AGT gene haplotype  had been reported to interact with I/D of the ACE gene [ AT1R gene in interacting with this AGT gene haplotype was not investigated. Because the six SNPs in the AGT gene are bound together due to its haplotype environment their potential interaction to SNPs of other genes may be limited. However, the joint effect of multiple SNPs between different genes may have a higher association degree than that of a single significant SNP. In the example of a natural data set with an unbalanced HT group and non-HT group, the unbalanced function based MDR algorithm is able to detect the significant association with hypertension in terms of 2- to 8-locus models by their OR and Boot-OR values  > T174M (2.810 − 2.372 = 0.438) > G-152A (3.241 − 2.810 = 0.431) > A-20C (3.863 − 3.241 = 0.622) > AT1R A1166C (4.510 − 3.863 = 0.647) > M235T (4.628 − 4.510 = 0.118).For hypertension association,R values . The hyp ACE I/D, suggesting that ACE I/D is mainly associated with hypertension. The G-217A and ACE I/D are integrated to the best 2-locus model. The G-6A is further integrated to the best 3-locus model and it is validated that G-6A has positively interacted with G-217A and ACE I/D (OR > 1). Similarly, the other newly integrated SNPs in each multiloci model have positively interacted with the previous SNPs in each multiloci model.The SNP-SNP interaction networks  have furMisclassification error is widely used as misclassification performance that aims to correctly estimate the proportions for an incorrect prediction. In MDR, the incorrect prediction error is an internal validation of a measurement that protects against finding chance associations in the sample. The misclassification errors of these multiloci models are much lower than 50%, indicating that the chance associations are significantly reduced.  AGT, ACE, and AT1R genes have an overall hypertension susceptibility effect. Among them, SNP I/D of ACE has the main association effect to hypertension and it also displays n-order interaction effect to SNPs of AGT and AT1R genes although they do not have a mutual effect on each other. The unbalanced function based MDR model can explore the epistasis network of SNPs AGT, ACE, and AT1R of RAS genes and identify strongly significant hypertension association. These interaction models and epistasis networks amongst AGT, ACE, and AT1R genes may be regarded as potential biomarkers for hypertension susceptibility. Our results also demonstrate that this powerful method has a potential to identify several disease-associated multiloci models as susceptibility biomarkers.In conclusion, hypertension is resulting from the interaction of several genetic risk factors. Analyses of multiple SNP-SNP interactions are complex and remain computational challenges when huge numbers of genetic factors are simultaneously considered. Moreover, the unbalanced data set may have to be analyzed with a bias due to the limitation nature of the MDR approach. In contrast, our proposed algorithm can constitute a nonparametric statistical analysis and provide a model-free and high-order-way measurement for epistasis without the limitation of a balanced data set. Accordingly, a significant outcome can be discovered from the high-order SNP-SNP interaction model amongst an unbalanced data set of many diseases including hypertension. Our results suggest thatAn illustrative example was provided to demonstrate how the unbalanced function based MDR works.


To evaluate the postoperative changes in blebs and levels of aqueous monocyte chemotactic protein-1 (MCP-1) after trabeculectomy vs. Ex-PRESS tube shunt surgery.Rabbits were subjected to trabeculectomy or Ex-PRESS tube shunt surgery and observed for up to 3 months. Intraocular pressure (IOP) was measured using a rebound tonometer. The MCP-1 level was measured by enzyme-linked immunosorbent assay (ELISA). Bleb morphology was evaluated using photos and anterior-segment optical coherence tomography (OCT).There were no differences in bleb appearance or IOP at any time between the groups. Bleb wall density in the anterior-segment OCT image was significantly lower 1 week after surgery in the Ex-PRESS group than the trabeculectomy group. The MCP-1 level in control eyes was 304.1 ± 45.2 pg/mL. In the trabeculectomy group, the mean aqueous MCP-1 level was 1444.9, 1914.3, 1899.8, 516.4, 398.3, 427.3, 609.5, 1612.7, 386.2, and 167.9 pg/mL at 3, 6, and 12 h, and 1, 2, 5, 7, 14, 30, and 90 days after surgery, respectively. In the Ex-PRESS group, the corresponding values were 1744.0, 1372.0, 932.5, 711.7, 396.1, 487.3, 799.5, 1327.9, 293.6, and 184.0 pg/mL. There were no significant differences in the aqueous MCP-1 level between the groups at any time point.The postoperative changes were similar in the Ex-PRESS and trabeculectomy groups, except for bleb wall density in the anterior-segment OCT image. The postoperative aqueous MCP-1 level had bimodal peaks in both groups. Trabeculectomy is the surgical method used most commonly to lower intraocular pressure (IOP) in glaucoma –2. RecenMonocyte chemotactic protein-1  is a potent chemotactic factor for monocytes that is involved in inflammatory cascades and fibrotic changes . MCP-1 iExperiments were conducted according to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Use Committee of Kumamoto University. The study used 126 female Japanese white rabbits . Sixty animals underwent trabeculectomy, another sixty underwent Ex-PRESS shunt surgery, and six animals were examined without surgery as controls. The animals subjected to surgery were euthanized after collecting aqueous humor 3, 6, and 12 h and 1, 2, 5, 7, 14, 30, and 90 days after surgery (n = 6 each) and the six control animals were euthanized without surgery. IOP was measured preoperatively and before collecting aqueous humor using TONOVET . The measurement was made before intramuscular injection of anesthesia.The animals were anesthetized with an intramuscular injection of ketamine hydrochloride  and xylazine hydrochloride . The right eye was washed with topical povidone iodine and a lid speculum was placed to hold the lids open. Then, a 7 mm conjunctival incision was made along the superior corneal limbus with spring micro-scissors to make a fornix-based conjunctival flap. A partial-thickness, trapezoidal-base, 3 mm scleral flap was created in the superior sclera, hinged at the limbus under the conjunctival flap. The scleral flap was dissected forward until it reached under the limbus. A sponge soaked with mitomycin C  was applied to the scleral flap and subconjunctival tissues for 3 min. Then, the MMC was washed out with normal saline . An ophthalmic straight knife  was inserted into the anterior chamber from underneath the scleral flap, creating an ostomy. The entrance to the anterior chamber was enlarged using a Kelly Descemet membrane punch . Forceps were used to pull the peripheral iris through the ostomy site and scissors were used to create the peripheral iridectomy. Using 10–0 nylon (MANI), two sutures were placed in the corner of the trapezoid that was not a hinge to close the scleral flap. Then the conjunctival peritomy was closed using 10–0 nylon sutures. At the end of the surgery, ofloxacin ointment  was applied. No additional medications were given postoperatively.The Ex-PRESS shunt devices were provided from Alcon Japan . The procedure was the same as the trabeculectomy described above until the MMC was washed out. Afterward, a 25-gauge needle  was inserted into the anterior chamber from underneath the scleral flap. Then the Ex-PRESS shunt device (Alcon Japan) was placed in the anterior chamber through the ostium created by the needle, following the manufacturer’s protocol. After fixation of the Ex-PRESS device, the scleral flap and conjunctival incision were sutured with 10–0 Nylon, as in the trabeculectomy described above. Ofloxacin ointment was also applied. No additional medications were given postoperatively.http://blebs.net/) by three independent researchers who were blind to the condition of the bleb .A photo was taken of each bleb with a digital camera , and its area and vascularity at 1, 2, 4, and 12 weeks postoperatively were classified using the Moorfields Bleb Grading System  2, 5, 7, and 14 days postoperatively , and the intensity of the bleb wall in the horizontal image was measured using CASIA bleb assessment software ver. 4.0L (Tomey).Anterior-segment OCT images were acquired using CASIAAqueous humor was collected from the rabbit eyes under anesthesia using the method described above. In total, 100–150 μL aqueous humor was gently withdrawn. Contact with the intraocular tissues was carefully avoided and contamination of the samples with blood was prevented. After centrifugation at 7830 g for 5 min at 4°C, a 50 μL sample was immediately stored at –80°C.The concentrations of MCP-1 in the aqueous humor of the rabbits were measured with an enzyme-linked immunosorbent assay . The absorbance was read at 450 nm in a microplate reader . The detection range was 15.6–1,000 pg/mL. To ensure that there was sufficient material for the experiment, the aqueous humor was diluted four times with phosphate-buffered saline .2O2 in methanol, the sections were treated with 10% normal goat serum for 30 min at room temperature (RT), and incubated with goat anti-α-SMA antibody  for 30 min at RT. For dual labeling, the sections were washed and incubated with affinity-purified, biotin-conjugated donkey anti-goat IgG secondary antibody  for 2 h at RT and then washed and incubated with detection reagents  for 30 min at RT. As a chromatogen, 3,3’-diamino-benzidine tetrahydrochloride  was used. Hematoxylin was used as a counterstain. As a negative control, adjacent sections were incubated without primary antibodies. The slides were observed using a fluorescence microscope , and some of the acquired photos were combined using software .Immunohistochemistry analysis has been described previously . BrieflyIOP and concentrations of MCP-1 were compared between the trabeculectomy and Ex-PRESS groups using the Wilcoxon rank-sum test. Postoperative IOP and concentrations of MCP-1 were analyzed using Dunnett’s test, using the preoperative/no-surgery values as respective controls. P-values less than 0.05 were considered significant.Representative blebs are shown in Generally, the amount of α-SMA positive cells represents the extent of tissue scarring at the time point. The immunohistochemical analysis showed immunoreactivity for α-SMA in cells around the fluid cavity in the bleb, suggesting that the conjunctiva cells partially transdifferentiated into myofibroblast-like cells . QualitaEx-PRESS tube shunt surgery has several advantages over conventional trabeculectomy. First, the device does not require a sclerostomy or peripheral iridectomy; therefore, it induces a smaller postoperative inflammatory reaction and causes less hyphema ,9,16–18.In the present study, the blebs in both study groups had the same appearance and tended to become flat within 3 months of surgery. One retrospective study reported that bleb height was lower in an Ex-PRESS group in the first 3 months postoperatively, and the blebs were more diffuse after 3 to 18 months compared to a trabeculectomy group . BecauseTo the best of our knowledge, no study has compared anterior-segment OCT images between Ex-PRESS tube shunt surgery and trabeculectomy. OCT provides more quantitative data on the internal structures of blebs than a slit-lamp examination, and is useful for understanding the postsurgical mechanisms of bleb failure. Our study compared anterior-segment OCT bleb images in a limited number of eyes over a limited period. Although our data are not conclusive, they suggest that there is a difference in bleb wall density between trabeculectomy and Ex-PRESS tube shunt surgery. Because the internal structures of blebs observed with anterior-segment OCT reflect future control of IOP –22, furtIn the present study, the IOP was lower until 1 month after surgery, and then it returned to the preoperative level in both groups. The time-dependent changes in the two groups were similar. In previous clinical studies, the postoperative IOP control and success rates were similar between Ex-PRESS tube shunt surgery and trabeculectomy ,16–19. S15 Therefore, the aqueous MCP-1 might be involved in the inflammatory response and scarring process after filtration surgery for glaucoma. Postoperatively, the time-course of the aqueous MCP-1 level showed bimodal peaks (at 3–12 h and 2 weeks) in both groups, which was unexpected. Previously, we measured the MCP-1 concentration after phacoemulsification in the aqueous humor of rabbit eyes, and found that the level 30 days after surgery was significantly higher than the preoperative level (207.1 ± 62.9 vs. 31.2 ± 12.5 pg/mL). In this study, a preliminary experiment revealed marked MCP-1 elevation 12 h after phacoemulsification, but the level returned to near basal levels within several days (data not shown), which represented a unimodal peak in the acute phase. As the first peak in the aqueous MCP-1 level was observed at a similar postoperative time in both eyes after filtration surgery and phacoemulsification, while the second peak 2 weeks after surgery occurred only in eyes after filtration surgery, different postoperative inflammatory reactions may have occurred in the anterior chamber in eyes after filtration surgery compared to after phacoemulsification.The chemotactic factor MCP-1 is involved in tissue infiltration of monocytes and T cells in a variety of inflammatory diseases. Previously, we reported that the aqueous MCP-1 level in phakic eyes with open-angle glaucoma predicted the results of trabeculectomy.The mechanism underlying the bimodal peak in the aqueous MCP-1 after filtration surgery is unclear. The general wound-healing process is divided into four phases: hemostasis, inflammation, proliferation, and remodeling . The firThe aqueous MCP-1 level was significantly higher 6 and 12 h after surgery in the trabeculectomy group than in non-operated eyes, whereas a significant difference was observed only at 3 h during the corresponding period after surgery in the Ex-PRESS group. Strikingly, the mean concentration at 12 h in the Ex-PRESS group was half of that in the trabeculectomy group (932.5 ± 531.2 vs. 1899.8 ± 515.0 pg/mL), although the difference was not significant (P = 0.109). This difference might partly explain the rapid recovery of vision after Ex-PRESS tube shunt surgery compared to trabeculectomy. Taken the difference in bleb-wall intensity between the groups into consideration, there might be small differences in the molecular mechanisms of the wound-healing process between the groups. Thus, though the post-surgical IOP was controlled similarly in this basic study and past clinical studies –19, the What subclinical mechanisms, if any, do offset the advantages of Ex-PRESS tube shunt surgery described above, including the time-dependent changes in the aqueous MCP-1 concentration, leading to the similar surgical success rates to trabeculectomy? One possibility is foreign-body reaction to the Ex-PRESS tube made of implantable stainless steel. However, the implantation of this device at the corneoscleral junction of the rabbit eye resulted in minimal inflammatory and scarring reactions , suggestThis study was limited by the relatively small sample size and the 3-month observation period. The fact that we studied rabbits must also be considered when discussing the clinical relevance of our results. In addition, the rabbits used in this study were not glaucoma model, and therefore the pathophysiological backgrounds of the rabbits were different compared to human surgical cases.In conclusion, the postoperative appearance of blebs, IOP control, and immunohistochemical findings were similar between the Ex-PRESS and trabeculectomy groups. The density of the bleb wall in the anterior-segment OCT images was significantly lower 1 week after surgery in the Ex-PRESS group compared to the trabeculectomy group. The postoperative aqueous MCP-1 level showed bimodal peaks in both groups, and there were no significant differences at any time point between the groups.


In contrast to other known l-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards l-GLN. In this study, some critical amino acids forming the active site of HpASNase  have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in l-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.Bacterial asparaginases  are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of  Helicobacter pyloril-asparaginase  is a type II l-asparaginase. The enzyme is capable of carrying out both l-asparagine (l-ASN) and l-glutamine (l-GLN) deamination with a stronger preference for the former. In contrast to other type II l-asparaginases, HpASNase was found to be cooperative and to have a lower affinity vs.l-GLN [l-asparaginases.s.l-GLN . Such chl-asparaginases, which are an important class of chemotherapeutic drugs [Bacterial ic drugs , are tetic drugs . Each suic drugs . The enzic drugs ,7,8. Theic drugs .l-asparaginases depends on two highly conserved catalytic triads consisting of Thr-Tyr-Glu and Thr-Asp-Lys [Helicobacter pyloril-asparaginase [The catalytic mechanism of -Asp-Lys ,10,11,12raginase ,11. Withl-asparaginases from different microbial sources show different kinetics towards the two substrates. Particularly, Helicobacter pylori CCUG 17874 l-asparaginase (HpASNase), differently from other type II ASNases, has a quite similar catalytic activity vs.l-ASN and l-GLN but an almost 150 times higher affinity for the former substrate [l-asparaginase with a cooperative kinetic vs.l-GLN. The explanation of such behavior can rely on structural differences between HpASNase and other type II l-asparaginases, most of all in intersubunits interactions at the intimate dimer interface. Analysis of the HpASNase highly homologous (92% sequence identity) Helicobacter pylori J99 l-asparaginase (HpA) structure highlights some specific peculiarities, mostly in the flexible loop (residues 19–46) and in the 286–297 loop, which may explain the HpASNase peculiar catalytic properties [Moreover, despite their highly similar structural architecture, type II operties .l-asparaginases (T16 and T95) and/or laying on structural elements that mostly differ from other l-asparaginases. In fact, residue E289 belongs to the 286–297 loop that in HpA is closer to the active site than in other type II l-asparaginases. Because of its position, E289 is involved in substrate binding and intersubunits interactions and therefore it can play a unique role in HpASNase catalytic properties.In this study, some critical amino acids forming the active site of HpASNase  have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Particularly, we focused on residues belonging to the active site that are highly conserved in l-asparaginases. Sequencing of the mutated insert revealed the presence of the desired replacement in all the cases except for a random replacement at position n.283 (C > G) resulting, instead of the planned T95D, in the amino acid replacement T95H. Beyond T95D, also the serendipitous T95H mutant was characterized. In fact, mutation T95H may strongly perturb the enzyme active center both directly and at long range, altering enzyme catalysis and/or substrates binding. Moreover, the T95H mutant structural model shows a delocalization of K168, a residue of the first catalytic triad (See Discussion Section).In our work we aimed at understanding the peculiar kinetic behavior of HpASNase by a deep analysis of its active site. In order to do this, we subjected the HpASNase gene to site direct mutagenesis to obtain T16E, T95D and E289A replacements. The replaced amino acids are part of the active site and, interestingly, E289, a subunits interface residue, belongs to a loop that differs for its spatial localization from other bacterial l-ASN and l-GLN. Their quaternary structure folding was verified by analytic gel-filtration and the profiles were not significantly different from wild type [l-ASN and with a 40-fold reduced kcat  and the Km was almost unchanged . In the case of T95H and T95D mutants, the presence of a larger or a charged residue, respectively, may compromise the proper catalytic triad organization, affecting mostly the K168 side chain localization B.l-asparaginases. Crystallographic evidences showed that in HpA the loop is closer to the active site, especially with respect to the EwA (Erwinia chrysanthemi type II l-asparaginase) enzyme [l-ASN (40-fold reduced kcat) and to be practically devoid of activity toward l-GLN. Interestingly, the Km value vs.l-ASN was almost unchanged, suggesting that the E289 residue has a marginal role in substrate binding, whereas its catalytic role and its intersubunit interactions are needed for proper catalysis in HpASNase, as hypothesized on the basis of structural data. Moreover, the dramatic loss of activities due to the E289 removal suggests that interactions between the 286–297 loop and the active site of HpASNase are essential in order to properly coordinate both substrate and catalytic residues, a feature not shared with other type II l-asparaginases (like EwA), where such interactions are absent [Residue E289 belongs to the 286–297 loop that is very variable in position in ) enzyme ,15. In t) enzyme . The HpAe absent .Although the E289A mutant presented a dramatically reduced catalytic activity, the enzyme resulted to be very thermostable. The naturally occurring E289 residue is solvent accessible and locates at the intimate dimer interface. Replacement of E289 with an alanine removes the canonical intersubunit ionic interactions and most likely gives rise to a new set of interactions leading to a more rigid and stable enzyme conformation. Moreover, this new conformation could be the reason of the inability of the mutant to perform the correct catalysis.H. pylori CCUG 17874 l-asparaginase, the pDC1 plasmid derived from pET101 D-TOPO plasmid  and containing the ansB wild type gene [PfuUltra buffer, 0.1 mM dNTPs, 0.5 µM each sense and anti-sense primers, 2.5 ng template dsDNA and 2.5 U PfxUltra (Invitrogen) in a final volume of 50 µL. PCR products were checked on a 1% agarose gel and digested with Dpn I restriction enzyme  at 37 °C for 1 h in order to remove all the native DNA before transformation. The oligonucleotides used as mutagenesis primers are reported in To obtain enzymatic variants of E. coli BL21(DE3) pLysS cells transformed with plasmid pDC1 mutant derivatives were grown in a ZYP-5052 medium 2SO4, 1 mM MgSO4, 0.5% w/v glycerol, 0.05 w/v glucose, 0.2% w/v α-lactose) containing 100 μg/mL ampicillin and protein expression was performed through auto-induction according to Studier [ Studier .et al. [Cells harvested from 1 L culture broth were suspended in 50 mM Na-phosphate pH 8.0, 300 mM NaCl, 10 mM imidazole (ImOH) added with 1 mM PMSF, sonicated and centrifuged. HpASNase recombinant variants were purified following the procedure used for the wild type enzyme [et al. .l-asparaginase and l-glutaminase activities were determined at 37 °C with l-ASN or l-GLN, respectively, as a substrate, by l-glutamate dehydrogenase coupled spectrophotometric assay according to Balcão [l-glutamate dehydrogenase  and 5 mM l-ASN or 150 mM l-GLN, in a final volume of 0.5 mL. The reaction was started by adding enzyme solution (5–50 μg) and NADH consumption was monitored at 340 nm with a Jasco V-550 UV/VIS spectrophotometer .Both o Balcão  and Derso Balcão  and in to Balcão . Brieflyl-ASN or l-GLN concentrations (0–10 mM l-ASN or 0–400 mM l-GLN). The reaction was started by adding the substrate to the reaction mixture containing 5–50 μg enzyme solution. The kinetic parameters Vmax and Km were determined both by Lineweaver-Burk and by Michaelis-Menten plots using the Enzyme Kinetic Module 1.1 for Sigma Plot .For steady-state kinetic studies, enzyme activity was assayed in triplicate under the above conditions using at least 10 different cat or turn-over number, is the number of catalytic events per second. Km is the substrate concentration at which the reaction speed is half maximal. kcat/Km is an index of catalytic efficiency at sub-saturating substrate concentration.kOne Unit is defined as the amount of enzyme needed to convert 1 µmole of substrate in product in the above described conditions.50. To evaluate the stability of HpASNase variants by T50, enzyme solutions (0.5–1.0 mg/mL) were incubated at different temperatures in 20 mM Na-phosphate pH 7.5 for 10 min. After incubation, the enzyme solution was chilled on ice and the activity was spectrophotometrically assayed. The residual activity was expressed as the percentage of activity of heat-treated enzyme with respect to the untreated one.A parameter useful to determine protein stability is THelicobacter pylori J99 structure (PDB ID: 2WLT) was used as a template. Modeling was performed using Modeler 9.13  [For structural modeling of T16E, T95D, T95H and E289A HpASNase variants the released CA, USA) .i.e., T16D and T95E [E. coli ASNase T89V mutant [vs.l-GLN. No information could be derived in this respect, as this residue was found to be strictly needed for catalytic activity vs.l-GLN. However, the loss of glutaminase activity together with the dramatic reduction in asparaginase activity, strongly suggest that the localization of the 286–297 loop in HpASNase near the active site allows for interactions which are required for the enzyme proper catalysis. This is the first time that kinetic data confirm the catalytic role of this structural element that differs among asparaginases. Further dissection of this feature could help in better understanding their different kinetic properties. A totally unexpected result lies in the increased thermal stability of the E289A mutant, which suggests a stabilizing role for this mutation and, in reverse, a potential role for destabilization in catalysis, a feature which would also deserve further investigation.In this work, we have investigated the role of three catalytic residues in HpASNase enzymatic activity. Our data demonstrate once more an essential role for both triad threonines T16 and T95 in asparaginase and glutaminase activity, a feature shared with all other members of the same class. The present data, in fact, resemble the characteristics of other two HpASNase mutants with amino acid replacement on the same residues (and T95E ), and thV mutant . Because


Compared to WT controls, sEH-deficient hearts required significantly greater basal coronary flow to maintain the perfusion pressure at 100 mmHg and exhibited a greater reduction in vascular resistance during tension-induced heart work, implying a better coronary perfusion during cardiac performance. Cardiac contractility, characterized by developed tension in response to changes in preload, was potentially increased in sEH-KO hearts, manifested by an enlarged magnitude at each step-wise increase in end-diastolic to peak-systolic tension. 14,15-EEZE (EET antagonist) prevented the adaptation of coronary circulation in sEH null hearts whereas responses in WT hearts were sensitive to the inhibition of NO. Cardiac expression of EET synthases (CYP2J2/2C29) was comparable in both genotypic mice whereas, levels of 14,15-, 11,12- and 8,9-EETs were significantly higher in sEH-KO hearts, accompanied with lower levels of DHETs. In conclusion, the elevation of cardiac EETs, as a function of sEH deficiency, plays key roles in the adaptation of coronary flow and cardiac function.Roles of soluble epoxide hydrolase (sEH), the enzyme responsible for hydrolysis of epoxyeicosatrienoic acids (EETs) to their diols (DHETs), in the coronary circulation and cardiac function remain unknown. We tested the hypothesis that compromising EET hydrolysis/degradation, via sEH deficiency, lowers the coronary resistance to promote cardiac perfusion and function. Hearts were isolated from wild type (WT), sEH knockout (KO) mice and WT mice chronically treated with  Following the acceptance of cyclooxygenase (COX) and lipoxygenase metabolites of arachidonic acid (AA) as clinical targets for the treatment of inflammatory disorders, the cytochrome P-450 (CYP/epoxygenase) enzyme has emerged as another noteworthy pathway for AA metabolism. AA metabolism by CYP/epoxygenase results in the production of epoxyeicosatrienoic acids (EETs), metabolites that have intrigued researchers and gained considerable attention due to their bioactive properties, which have been shown to be involved in cellular signaling and cardiovascular protection  and Ephx2+/+  mice were used. As described previously  and WT mice were received from the Jackson laboratory  and the homozygous (sEH-KO) mice were developed in the Department of Comparative Medicine, New York Medical College. One group of WT mice received t-TUCB , via oral gavage, for 4 weeks.Twelve- to fifteen-week-old male Ephx2All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the guidelines of the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals.t-TUCB-treated mice, changes in blood pressure were monitored twice per week by tail-cuff blood pressure measurements during the course of treatment, as well as by carotid artery catheterization on the day the experiment was conducted.Mice were anesthetized by inhalation of Isothesia (isoflurane). Blood pressure was recorded using carotid artery catheterization and a flow-through pressure transducer . The heart rate of anesthetized mice was controlled at approximately 480 beats per minute by adjusting the depth of anesthesia when the blood pressure was measured. In 4 1.1, KH2PO4 1.2, glucose 5.5, CaCl2 2.5, pyruvate 2.0, ascorbate 0.1, L-arginine 0.1, and NaHCO3 24. The PSS was gassed in a water-jacketed (37°C) reservoir with 95%O2–5% CO2 to a pH value of 7.4. The perfusate was pumped through an in-line 1 μm filter using a Rainin peristaltic pump. A 2-mL bubble trap, a water-jacketed (37°C) heating coil and a T-adaptor connecting a pressure transducer  were sequentially connected to the heart. A metal hook made from a 31 gauge needle was penetrated through the left ventricle at the apex of the heart to drain the Thebesian fluid accumulated in the ventricle. The other end of the hook was attached to a force transducer  to control and record tension and heart rate. The heart was immersed in the coronary effluent solution in a water-jacketed (37°C) tissue bath . The heart was perfused with a constant flow. Changes in perfusion pressure, end-diastolic, and peak-systolic tension were continuously recorded. At the beginning of experiments, the flow rate was rapidly increased to reach a perfusion pressure of 100 mmHg. During the equilibration, changes (increases) in perfusion pressure as a consequence of development of myogenic constriction in coronary arteries were continuously adjusted by reducing flow rate to maintain a perfusate pressure at 100 mmHg. The hearts reached an equilibrated status (a constant perfusion pressure of 100 mmHg and a constant heart rate of above 320 beats per min) within 15 min. The flow rate provided to obtain a constant perfusion pressure of 100 mmHg was considered as basal coronary flow and was kept constant throughout experiments. After basal flow was obtained, tension was increased from 0 to 5 g in 0.5-g increments to initiate a step-wise increase in end-diastolic tension of the heart. At each tension step, changes in perfusion pressure were continuously recorded for 5 min on a DI-700 data acquisition system .As described previously  or 14,15-epoxyeicosa-5(Z)-enoic acid , or L-NAME plus EEZE were added into the PSS. Hearts were perfused with inhibitor-containing PSS for 45 min before recording the basal flow, and then changes in perfusion pressure in responses to stepwise-increases in tension were recorded.In separate groups of mice, NAfter perfusion experiments, hearts were dabbed on paper towel to remove excessive fluid and weighted to obtain the wet weight of hearts. Hearts were then wrapped with aluminum foil and placed in an 80°C oven for at least 48 h until a constant dried weight obtained.Isolated hearts were pulverized in liquid nitrogen. Phospholipids were extracted with the Bligh-Dyer method from 30 to 50 mg of heart tissue. EETs and DHETs were extracted with ethyl acetate following alkali hydrolysis of the phospholipids to release esterified EETs or DHETs and quantified with a Q-trap 3200 linear ion trap quadruple LC/MS/MS  equipped with a Turbo V ion source operated in negative electrospray mode , as described previously  extracted from hearts were loaded on and separated by a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was probed with specific primary antibodies for sEH, eNOS, CYP2J2 , and CYP2C29 , respectively, and appropriate secondary antibodies conjugated with horseradish peroxidase. Specific bands were visualized with a chemiluminescence kit and normalized to GAPDH. The X-ray film was scanned into a computer and band densitometry was digitalized with UN-SCAN-IT software.Isolated hearts were pulverized in liquid Nt-test or pair t-test where appropriate. Statistical significance was accepted at a level of P < 0.05.Data are expressed as means ± SEM and n refers to the number of mice. Basal coronary flow was recorded at a constant pressure of 100 mmHg and normalized to heart weight, and expressed as mL/min/g. Coronary vascular resistance was calculated by the formula of R = P/F, where P and F are the indicative of perfusion pressure and flow, respectively, and expressed as mmHg/min/mL. Analyses of linear regression and the comparison of the slopes of regression lines between multiple groups were performed using GraphPad Prism software . Analyses of animal and heart parameters, coronary basal flow and cardiac level of metabolites were performed using Student's t-TUCB for 4 weeks. Blood pressure including systolic, diastolic, and mean arterial pressure, was significantly reduced in mice with the sEH gene genetically knocked down or sEH activity pharmacologically inhibited as compared to their respective WT controls. Body weight (BW) was comparable among the three groups, but heart weight  was significantly increased in sEH-KO compared to WT mice, leading to a greater ratio of HW/BW in sEH-KO mice. The ratio of HW/BW between t-TUCB-treated WT and WT controls was comparable.TableThe functional consequence of Ephx2 gene deletion was documented by LC/MS/MS analysis. TableFigurex-axis) received by the heart to a group of relative numbers. Given that the normalization does not alter the statistical significance, but has an unnecessary impact on the expression of data, the analysis of the data without normalization is therefore, preferred over the normalized data.Next, changes in perfusion pressure expressed as changes in vascular resistance, were recorded in response to step-wise increases in end-diastolic tension in hearts of WT and sEH-KO mice. As depicted in Fig.Developed tension, represented as a difference between peak-systolic and end-diastolic tension, was compared between WT and sEH-KO hearts in response to increases in end-diastolic tension. As shown in Fig.On the basis of aforementioned findings, we next aimed to elucidate underlying mechanisms responsible for the enhanced coronary flow and cardiac contraction in sEH-KO mice. The correlation of cardiac work, which was quantitated by the double product (developed tension × heart rate), and coronary resistance was compared between the two groups. FigureFiguret-TUCB (an inhibitor of sEH) for 4 weeks. Figuret-TUCB resulted in a downward-shifted regression line in the end-diastolic vs. resistance curve. The rate of reduction in coronary resistance was significantly greater in t-TUCB-treated than that of control mice, as evidenced by a deeper slope.In separate experiments, sEH-dependent modulation of the coronary circulation was evaluated in WT mice that had been treated with This study demonstrates the specific role of deleting the sEH gene or pharmacological inhibition of sEH enzyme in the adaptation of coronary circulation, characterized as enhanced coronary flow Fig. and attet-TUCB. Both groups of mice exhibit a similar responsive phenotype . Essentially, regardless of the type of normalization used, the statistical significance between the two strains of hearts was constantly maintained. Also, our aim in using Langendorff-perfused heart was to observe and evaluate cardiac function comprehensively as a whole, via an isolated organ. Therefore, on the basis of these concerns, we have presented most of our data in this study without normalization.SHH and BDH are the authors of UC Patents on SEHI chemistry and blood pressure regulation.


There is much interest in confirming whether the efficacy of abiraterone acetate (AA) demonstrated within the trial setting is reproducible in routine clinical practice. We report the clinical outcome of metastatic castration-resistant prostate cancer (mCRPC) patients treated with AA in real-life clinical practice.The clinical records of mCRPC patients treated with AA from all 6 public oncology centers in Hong Kong between August 2011 and December 2014 were reviewed. The treatment efficacy and its determinants, and toxicities were determined.p = 0.0007) and PFS  than those without. Pain control was comparable in both groups of patients. The most common grade 3 or above toxicities were hypertension (6.9/5.8 %) and hypokalemia (3.4/3.8 %) in chemo-naive/post-chemo patients. In multivariate analysis, the presence of prostate-specific antigen (PSA) response (≥50 % drop of PSA from baseline) within the first 3 months of therapy was associated with favorable OS and PFS in both chemo-naive and post-chemo group.A total of 110 patients with mCRPC were treated with AA in the review period, of whom 58 were chemo-naive and 52 had received prior chemotherapy (post-chemo). The median follow-up time was 7.5/11.4 months for chemo-naive/post-chemo patients. 6.9/15.4 % of chemo-naive/post-chemo patients had visceral metastases. The median overall survival (OS) and progression-free survival (PFS) were 18.1/15.5 months and 6.7/6.4 months for chemo-naive/post-chemo patients, respectively. Among chemo-naive patients, those with visceral diseases had significantly inferior OS  was considerably shorter than that reported in the COU-AA-302 trial (34.7 months), and the OS was particularly short in those with visceral metastases (2.8 months). Conversely, AA was efficacious in post-chemo patients. AA resulted in comparable pain control in both groups of patients. The presence of PSA response within the first 3 months of treatment was a significant determinant of survival. Androgen deprivation therapy (ADT), either medical or surgical, is the backbone of first line treatment for metastatic prostate cancer . While uSince 2004, docetaxel chemotherapy was the standard of care for patients with mCRPC , 4. MoreThere is much interest in confirming whether the efficacy of AA demonstrated within the trial setting is reproducible in routine clinical practice, in consideration of possible differences in selection of patients, ethnic differences, and other factors in day-to-day practice. In fact, for the case of docetaxel for mCRPC patients, previous retrospective studies had shown unexpectedly higher incidence of febrile neutropenia and less favorable survival outcome compared to that in the trial setting , 9. In tThe study was approved by the institutional review board of the authors’ institutions . And permission to access the medical records through the inter-hospital computer network was granted by the aforementioned review board. Furthermore, the principles of the Helsinki Declaration were followed. Informed consent has been exempted by the review board as most of the patients in this study were dead when the data was collected.In early 2011, AA was approved by the local health authority for use in patients with mCRPC who had received prior chemotherapy, and subsequently in 2012, also for chemo-naïve patients. The present review included mCRPC patients who were started on AA in 6 oncology centers between August 2011 and December 2014. All patients had metastatic prostate cancer which had progressed despite achieving castration-level of testosterone. Enzalutamide, another AR pathway targeted agent, was not accessible during the study period and only be commercially available since October 2015 in our locality . PatientThe electronic clinical records of the patient cohort were retrieved by the inter-hospital computer network. The definition of clinical, biochemical and radiological progressive disease was according to the Prostate Cancer Clinical Trials Working Group (PCWG-2) criteria . OverallP values ≤ 0.05 were considered significant. The hazard ratio (HR) and the corresponding 95 % confidence interval were calculated.Statistical analysis was performed using the Statistical Package for the Social Sciences . The updated database as at 1 May 2015 was used for analysis. Kaplan-Meier plots of OS and PFS were obtained for subsets of patients segregated by various potential prognosticators. The log-rank test was employed to assess the difference in outcome between subsets. The variables were also subject to multivariate analyses using the Cox proportional hazards regression model. Hundred and ten patients were reviewed, of whom 58 were chemo-naïve and 52 were post-chemo. Table The proportion of patients with PSA response , PSA flare (about 30 % of patients), and eventual response after PSA flare (about two-thirds of patients with flare) were similar between the chemo-naïve and post-chemo groups Table . All of The median duration of AA treatment was 6.8  and 7.1 (0.5–25.0) months for chemo-naïve and post-chemo group respectively, with 27 chemo-naïve, and 13 post-chemo patients still under treatment at the time of last follow-up. Disease progression was the major reason of treatment discontinuation . In contrast, the differences in OS and PFS were not significant between patients with or without visceral disease in the post-chemo group : 9.9–25) and 15.5 (95 % CI: 13.8–23.6) months for chemo-naïve and post-chemo group respectively , were significantly determinants of the OS (Table p = 0.0015), presence of PSA response , short (<10 months) response to prior ADT , ECOG 2 or above , and low hemoglobin level  were determinants of OS. Presence of PSA response  and presence of visceral disease  were determinants of PFS.In univariate analysis, 6 variables, including the presence of visceral disease , Gleason score of ≥ 8  and PSA-DT of < 2 months  were determinants of OS, while presence of PSA response  was a determinant of PFS  was associated with an unfavorable survival in chemo-naïve patients with AA in this study. Our finding substantiates the other reports that the short response to ADT was associated with poorer efficacy with AR-pathway targeted therapy, in particular AA, in mCRPC patients , 17. AndLimitations existed in the present study, which include the typical shortcomings of retrospective study such as under-reporting of adverse events, incompleteness of data collection and selection bias etc. However, we consider these limitations would not affect the ability to capture the survival outcome of AA in this study. And the inadequate sample size, difference in follow-up protocols and the policy of post-AA treatment among different hospitals were the other weakness of the current study. Of note, unlike prospective study, regular imaging was not mandatory in our study and this could deprive some patients from other life-prolonging treatment earlier before any clinical or biochemical progression existed. Finally, the follow-up time for the chemo-naive group is comparatively inadequate and the inferior outcome in this group may not be the ultimate result. Our group will plan for another follow-up study in the future.The present study reported the unanticipated short survival after AA in chemo-naïve patient outside clinical trial setting. The overall survival was particularly short in those with visceral diseases, and further clinical trial for AA in this subgroup of patients is warranted. In contrast, AA was well tolerated and efficacious in mCRPC patients with prior chemotherapy. AA resulted in comparable pain control in both groups of patients. PSA response, in particular present within the first 3 months after AA, could serve as a prognostic biomarker for survival outcome and may have a potential role in selecting patients for additional or alternative treatment earlier in future clinical trial.The study was approved by the institutional review board of the authors’ institutions . Informed consent has been exempted by the review board as most of the patients in this study were dead when the data was collected.The dataset supporting the conclusion of this article is included within the article.